The present invention relates generally to sulfur oxide detection and more particularly to systems, methods, and materials for fluorometric detection and quantification of sulfur dioxide in a fluid sample. As used herein, the term "sulfur oxide" includes gaseous sulfur dioxide and its aqueous solutions, as well as sulfurous acid and its salts, but does not include sulfur trioxide, sulfuric acid, or its salts.
The instrumental methods utilized in the analysis of sulfur dioxide can be broken down according to four basic detection principles: colorimetry, conductivity, coulometry and flame photometry. (See, e.g., Coloff, S. G., et al., Amer. Lab., 5, pp. 10-22 (July, 1973).) Additionally, U.S. Pat. No. 3,659,100 relates to the use of the luminescent material, 5-amino-2,3-dihydro-1,4-phthalazinedione, commonly known as "luminol".
The most common of presently utilized methods employ colorimetric techniques and numerous analytical schemes involving such techniques have been suggested. U.S. Pat. No. 3,567,392, for example, relates to the use of a p-aminophenylazobenzene dye.
In 1971 the United States Environmental Protection Agency established national ambient air quality standards for major air pollutants including sulfur dioxide (Federal Register Vol. 36, No. 84, pages 8186-8201). The E.P.A. standards include a "reference" analytical method for sulfur dioxide determination [generally according to West, P. W., et al., Anal. Chem., 28, pp. 1816-19, (1956), as modified according to Scarangelli, F. P., et al., Anal. Chem., 39, pp. 1709-1719 (1967)]. The method involves collection and absorption of sulfur dioxide in sodium or potassium tetrachloromercurate (TCM) to form a dichlorosulfitomercurate solution. The absorbed sample is reacted in an analytical system with formaldehyde and a free amino-containing dyestuff, para-rosaniline. Spectrophotometric analysis of the resulting para-rosaniline methylsulfonic acid permits quantification of "trapped" sulfur dioxide. According to this method, concentrations of sulfur dioxide in the range of 26 to 1,050 .mu.g/m.sup.3 (0.01 to 0.40 parts per million) may ordinarily be measured without sampling very large volumes of air. The lower limit of detection is approximately 0.05 .mu.g./ml. of sulfur dioxide in the TCM trapping solution.
The use of the para-rosaniline method is by no means free of problems. It has been noted, for example, that multiple reactions occur between the para-rosaniline (which has multiple free amino sites) and the formaldehyde-bisulfite complex which is an intermediate in the Schiff reaction method. Further, it has been noted that many para-rosaniline samples obtained from various manufacturers contain impurities which may lead to analytical errors. Stock solutions of the para-rosaniline reagent ordinarily must be made up three days in advance of use. For adequate reproducibility, a closely monitored temperature of 45.degree.C. and a 30 minute development time is recommended.
In an attempt to avoid the apparently insoluble problems associated with the reference spectrophotometric method, numerous variant techniques have been proposed. Axelrod, H. D., et al., Anal. Chem., 42, pp. 512-515 (1970), for example, suggests the use of a similar Schiff reaction within a more sensitive fluorometric analytical procedure according to which the inherent fluorescence of 5-aminofluorescein is quenched in the course of a reaction with a formaldehyde-bisulfite complex. This method too has a rather long recommended development time. Employing a 5 .times. 10.sup.-.sup.4 M fluorescein reagent, the method has a lower detection limit of approximately 0.03 .mu.g./ml. sulfur dioxide in a TCM trapping solution. With the fluorometer reagent blank reading set at 100, such a "lower limit" reaction mixture gives a reading of about 98 after 15 minutes of development. As such, the method is less than optimally satisfactory in rapid analysis of multiple samples which differ in sulfur dioxide content by small amounts.
To date, no direct fluorometric sulfur oxide measurement scheme has been elucidated wherein a relatively non-fluorescent reagent proportionally reacts with the oxide to form quickly and easily measurable amounts of a fluorescent moiety.